Topical administration of toremifene and its metabolites

ABSTRACT

Transdermal preparations containing as active ingredient toremifene or one of its metabolites N-demethyltoremifene or 4-hydroxy-toremifene or their pharmaceutically acceptable non-toxic salts are useful for the treatment of cancers localized in the skin or on a short distance form the skin such as metastatic lesions of breast cancer. They may also be used in adjuvant therapy of breast cancer and in the reversal of multidrug resistance of cancer cells to cytotoxic drugs. Such transdernal preparations are of particular interest in the treatment of melanoma, lymphoma, Kaposi&#39;s sarcoma and fungoides mycosis.

This invention relates to topical preparations containing as activeingredient toremifene or one of its metabolites N-demethyltoremifene(4-chloro-1,2-diphenyl-[4-[2-(N-methylamino)ethoxy-]phenyl]-1-butene) or4-hydroxytoremifene (4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-[4-[2-(N,N-dimethylamino)ethoyoxy]phenyl]-1-butene) or their pharmaceuticallyacceptable non-toxic salts. The use of such topical preparations e.g.for the treatment of cancers localized in the skin or on a shortdistance from the skin such as metastatic lesions of breast cancer isalso within the scope of the invention. Moreover, the use of thesetopical preparations for the adjuvant therapy of breast cancer as wellas their use for the reversal of multidrug resistance of cancer cells tocytotoxic drugs are also within the scope of the invention. Topicaladministration of toremifene or its metabolites are of particularinterest in the treatment of melanoma, lymphoma, Kaposi's sarcoma andfungoides mycosis.

Tamoxifen and toremifene are triphenylethylene antiestrogens used in thetreatment of estrogen receptor positive breast cancer. These drugs arethe most frequently prescribed as endocrine agent for the treatment ofbreast cancer. Tamoxifen and toremifene inhibit estrogen-induced growthby competitive antagonism of tumor estrogen receptors (ER's).Antiestrogen therapy is effective in prolonging a disease-free state andoverall survival of women following primary surgery. Antiestrogentherapy delays recurrence and prolongs survival in patients with primarybreast cancer undergoing adjuvant therapy after mastectomy. Abouttwo-thirds of patients with estrogen receptor (ER) positive metastaticbreast cancer will have a temporary remission on tamoxifen. Althoughtamoxifen is considered a relatively benign drug, recent evidencesuggests that women receiving tamoxifen as adjuvant therapy may haveincreased risk of developing endometrial neoplasms (Fornander T et al,Lancet 1989, 21:117-120). Histopathology identifies these tumors asinfiltrating endometrial tumors unrelated to breast tumor metastasis.The factors contributing to this increased risk are not well understood.However, a variety of studies have linked endometrial cancer to agentswith estrogen activity (Smith Dc et al, N England J Med 1975; 293:1164-67).

The development of multidrug resistance (MDR) is one of the majormechanisms by which cancer becomes refractory to chemotherapeuticagents, especially anthracyclines and the vinca alkaloids. Classical MDRis associated with the overexpression of a mdr-1 gene that codes for aplasma membrane P-glycoprotein (p170). The expression of the MDR-1 geneis believed to be associated with a decreased cellular accumulation ofdrug due to an active dependent efflux mechanism.

Although many agents including verapamil, the trifluoperizines, andcyclosporins have been shown to reverse multidrug resistance in vitro,most agents do not achieve high enough in vivo concentrations to reversewithout substantial toxicity to the patient. This is particularly truefor verapamil which is associated with significant cardiotoxicity.

The non-steroidal triphenylethylene antiestrogens have demonstrated invitro chemosensitizing activity apparently unrelated to theirantiestrogenic effects. Toremifene and its metabolitesN-demethyltoremifene and 4-hydroxytoremifene are examples of newtriphenylethylenes that have chemosensitizing activity in MDR-positivecells at concentrations that are achieved in humans without significanttoxicity. Toremifene appears to be unique in that concentrations thatreverse resistance in vitro (5 μM) can be achieved in vivo followingoral therapy without substantial toxicity (Wiebe VJ et al, CancerChemother Pharmacol, 1990, 25: 247-251). Plasma concentrations oftoremifene and N-demethyl-toremifene following large oral doses are onthe order of 10 μM. However, plasma concentrations may not reflect theeffective anti-MDR activity at the tumor level. Although the systemictoxicity of high-dose toremifene (400 mg/day) is generally confined tovertigo, nausea and hot flashes, combination therapy with othercytotoxic agents may alter the systemic toxicity profile.

Therefore, methods of increasing the tumor concentration of toremifeneor its metabolites while minimizing systemic exposure may improve theefficacy of anti-MDR therapy with these drugs.

It has now been found that topical administration of toremifene and itsmetabolites can be used in order to achieve very high local tumorconcentrations without the risk of systemic toxicities. Such topicalpreparations of toremifene and its metabolites are useful in thetreatment of cancers localized in the skin or on a short distance fromthe skin, such as melanoma, lymphoma, Kaposi's sarcoma, fungoidesmycosis and localized metastatic lesions of breast cancer. In addition,topical administration may lead to an effective method of preventing therecurrence of breast cancer in high-risk patients while minimizing therisk of endometrial tumors and systemic toxicity.

The transdermal administration of toremifene and its metabolites can beaccomplished mainly in two different ways: (i) by mixing thetherapeutically active compound or its non-toxic pharmaceuticallyacceptable salt with suitable pharmaceutical carriers and optionallypenetration enhancers to form ointments, emulsions, lotions, solutions,creams, gels or the like, where preferably an amount of said preparationis applied onto a certain area of skin, or (ii) by incorporating thetherapeutically active substance into patches or transdermal deliverysystems according to known technology.

Examples of suitable excipients include those well known in the art ofpharmacy for the preparation of topical formulations such as DMSO,vegetable and animal oils, non-volatile fatty alcohols, acids, esters,e.g. cetostearyl alcohol and cetyl alcohol; volatile alcoholiccompounds, e.g. ethanol or isopropanol; glycols and glycol ethers,polyethylene glycol, polypropylene glycol, glycerol and glycerol ethers,cellulose derivatives, e.g. methylcellulose or carboxymethyl cellulose.Emulsifying agents, e.g. sorbitan stearate or polysorbate 60, andpreserving agents and penetration enhancers known in the art may also beincluded.

The dissolution properties into aqueous media of toremifene and itsmetabolites can be significantly improved by complexation of the drugsubstance with cyclodextrins. Cyclodextrins (including alpha, beta andgamma cyclodextrins and their derivatives) are all cyclic oligomers ofglucose. The cyclodextrins can form inclusion complexes with drugs inthat drug molecule is included in the lipophile-seeking cavities of thecyclodextrin molecule. Therefore the cyclodextrins effectivelysolubilize lipophilic drugs into aqueous media. The use of cyclodextrinsin the pharmaceutical field has been described e.g. in Drug Developmentand Industrial Pharmacy, 17(11), 1503-1549, 1991.

Dose range of toremifene or its metabolites for prevention/adjuvanttherapy is in the order of 1-200 mg/day/person in humans depending onthe bioavailability of the drug. The preferred dose would be close toadjuvant oral dose of 60 mg/day, or a single weekly dose of 300-500mg/patch. For high-dose short course toremifene (or toremifenemetabolite) a dose of 400-1000 mg/day is suggested; preferably 500-600mg/day.

EXPERIMENTS

Following extensive studies were carried out to substantiate the presentinvention:

Experiment 1 describes tissue distribution of toremifene and itsmetabolites following short course high-dose topical vs. intraperitoneal(IP) administration in the mouse, as well as tissue distribution ofantiestrogenic doses of toremifene and its metabolites following singledose topical vs, IP administration.

Experiment 2 describes efficacy of toremifene and its metabolitesfollowing topical administration in preventing breast cancer tumorgrowth in the mouse.

Experiment 3 describes efficacy of high-dose topically administeredtoremifene in enhancing doxorubicin cytotoxicity in MDR tumors in themouse.

Experiment 4 describes efficacy of toremifene and its metabolitesfollowing topical adminstration in preventing lymphoma growth in thebaboon.

Experiment 5 describes tissue distribution of toremifene followingtopical administration in Monodelphis domestica and the cytostaticeffect of toremifene in melanoma cell lines.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings,

FIG. 1 shows the results of cloning studies performed on the tumorsexcised from mice treated with topical and intraperitoneal toremifene,

FIG. 2 shows the results of cloning studies performed on tumors excisedfrom mice treated with and without topical toremifene,

FIG. 3a shows the response observed in baboon right inguinal lymphomatreated with topical toremifene, and

FIG. 3b shows the response observed in the untreated baboon leftlymphoma.

EXPERIMENT 1

Methods:

Sixteen female athymic nude mice were injected with a 20-gauge needlesubcutaneously with 5×10⁶ MDA A-1 cells in the left scapular area. Thetumors were allowed to grow for three weeks prior to treatment.

For IP administration toremifene citrate was suspended in peanut oil.Each of the three mice were injected IP using an 18 gauge needle with 50μl, delivering 1 mg/day for five days. Topical toremifene wasadministered in a mixture of DMSO/ETOH/methylcellulose/water(10:25:7:58) at a dose of 2.5 mg/animal/day for 5 days. Toremifenecitrate was first dissolved in DMSO and then diluted in the mixture ofETOH, methylcellulose and water. All mice were sacrificed on the fifthday of treatment two hours after the final dose. Blood specimens werecollected by capillary pipette after disruption of axillary vessels andplaced into heparinized centrifuge tubes. The brains, livers, and uteruswere removed surgically and placed in separate tubes.

Single dose toremifene was administered by IP as described above, ortopically with DMSO/ETOH/methylcellulose/water as the diluents. The doseof IP or topical toremifene was 500 μg. Tissue samples were collected asabove.

All blood samples were centrifuged at 1,000 g. The plasma was collected,measured for volume, and placed in clean extraction tubes. All remainingtissue samples were weighed, placed in extraction tubes, and spiked withan internal standard, nafoxidine. The tissues were thoroughlyhomogenized before the addition of 6 ml extraction fluid. Samples werecollected and dried as described below. Before injection onto the HPLCcolumn, each sample was reconstituted with 200 μl methanol. All sampleswere placed in a quartz cuvette for UV activation. The brain samplesrequired filtration with disposable syringe filters. Concentrations oftoremifene was calculated for all samples based on the standard curve.

Toremifene and its metabolites were quantified by high performanceliquid chromatography (HPLC).

Results

Table 1 shows that topically treated tumors had 150 times greaterconcentrations of toremifene while having a lower serum concentration at2.5 times the dose of intraperitoneal administration. This indicated aclear pharmacologic advantage. Interestingly, the mice receiving topicaltoremifene had one-tenth the concentrations in the uterus compared tothose receiving IP toremifene. In the liver, the topically treated micealso showed lower toremifene concentrations suggesting less systemicdistribution. The toremifene concentrations in the brain tissue wereequivalent. Table 2 shows that the topically treated tumors had agreater concentration of N-demethyltoremifene while having a lower serumconcentration. This indicates that MDA A-1 tumor cells may metabolizetoremifene. There was essentially no difference in concentration in theliver between the intraperitoneal and topically treated tumors. Thebrains and uteri of the topically treated mice also showed lessN-demethyltoremifene concentration than the IP treated group.

Table 3 shows that the topically treated tumors had greaterconcentrations of trans 4-hydroxytoremifene than those treated IP. Thismetabolite is the most potent antiestrogenic metabolite. Serum levels inthe topically treated tumors were lower, while in the brain,concentrations were identical.

We also examined the in vitro uptake of toremifene in the MCF-7 ER+cellsat effective antiestrogenic drug doses and compared these studies withthe results from the in vivo distribution studies described above.Following 20 hour incubation with 6.6 μM toremifene, cells were washed,counted, weighed and cellular toremifene concentrations were measured byHPLC. The concentration of toremifene (μg/mg) is shown in Table 4.

By comparison to toremifene concentrations achieved in tumors followingtopical and intraperitoneal routes of administration, the largestconcentrations are seen following topical administration of toremifene.Topical administration resulted in greater than 7 times the toremifeneconcentration found after in vitro exposure and greater than 150 timesthat found following intraperitoneal injection. These results suggestthat the concentrations of toremifene can easily be obtained followingtopical administration to subcutaneous tumors whereas intraperitonealadministration was 1/20 of the in vitro uptake data, thus suggesting afurther kinetic advantage of topical therapy.

Table 5 shows the toremifene concentrations following a single dose of500 μg IP vs 500 μg administered topically. DMSO and methylcellulosewere used as the diluents. Both topically administered methods hadsimilar tumor levels compared to IP but had much less systemicdistribution. This was trend noted in both serum and uterus.

Discussion:

In the experiment, we demonstrate that toremifene readily undergoestransdermal penetration and achieves very high tumor concentrationswhile limiting systemic exposure. Interestingly, while more toremifenecould be delivered topically to the tumors, the serum concentrationswere much lower indicating a significant therapeutic advantage fortopical delivery of toremifene. In addition, both the active N-demethyl-and 4-hydroxy-metabolites of toremifene were present in greaterconcentrations in tumor tissue following topical delivery than IPsuggesting that metabolism to these active metabolites may also occur atthe tissue level rather than exclusively in the liver.

The uterus and liver had much lower toremifene concentrations aftertopical delivery than IP providing further evidence of reduced systemicexposure with topical therapy. Reduced uterine exposure may be asignificant clinical advantage due to the potential risk of secondarytumors to the antiestrogens. Following prolonged adjuvant treatment withtamoxifen, endometrial dysplasia, bleeding, polyp formation and tumorshave been reported (Nuovo MA et al Int J Gyn Pathol 1989; 8: 125-131).Tamoxifen metabolites (Bisphenol and metabolite E) following tamoxifentherapy that are present in uterine tissue may potentially be associatedwith the development of secondary uterine neoplasms (Nuovo MA et al, IntJ Gyn Pathol 1989; 21: 117-120). Topical administration of toremifenemay circumvent the potential for these side effects.

EXPERIMENT 2

Methods:

In this study 12 mice were transplanted with MCF-7 cells and wereallowed to grow until a small tumor was detected in all mice (<1 mm³).At that time mice were divided into three groups of four mice each. Onegroup received the diluent alone (DMSO, 20 μl) topically, the secondgroup received 500 μg of 4-OH-toremifene by topical administration, andthe third received 500 μg/day of toremifene by topical administration.All mice received the same amount of diluent/day (20 μl ). The study wasassessed following 15 days of treatment.

Results:

All four control mice had developed tumors (>6×8 mms). In the othergroups only one mouse in the 4-OH-toremifene group developed a smalltumor (<2×1 mms). This experiment clearly shows that topicaladministration of 4-OH-toremifene or toremifene prevented tumor growth.

Discussion:

This experiment clearly demonstrates that adjuvant doses of topicallyadministered toremifene or its metabolites can prevent breast cancertumor growth in an in vivo mouse model. This result coupled with thekinetic advantage of topical administration (Experiment 1) suggests thattopical toremifene can achieve the desired antiestrogenic effectswithout systemic distribution.

EXPERIMENT 3

Methods:

The topical administration toremifene citrate was dissolved in 1.0 ml ofdimethyl sulfoxide (DMSO) resulting in a solution delivering 2.5 mgtoremifene/60 μl. Drug was applied dropwise to the skin surface directlyand around the tumor. Each drop was allowed to evaporate completely.This treatment was administered once a day at the same time for a totalof five days. All mice were sacrificed on the fifth day of treatment twohours after the final dose. Tumors were collected sterilely andprocessed for clonogenic assay.

Following toremifene administration tumors were excised and a portion ofeach tumor was extracted for HPLC analysis. The remaining portion wascloned following a 1 hour exposure to doxorubicin 1 μg/ml. The percentinhibition was determined after 14 days, and plotted against thetoremifene concentration in each individual tumor.

Results:

FIG. 1 shows the results of cloning studies performed on the tumorsexcised from mice treated with topical and intraperitoneal toremifene.As shown in the graph there was a good correlation between percentinhibition and cellular toremifene concentration at 14 days (r=0.77). Inaddition, all three topically treated tumors achieved >30% inhibition,whereas all three intraperitoneal treated tumors had <30% inhibition.

FIG. 2 shows the results of cloning studies performed on tumors excisedfrom mice treated with and without topical toremifene. The top curveshow a tumor resistant to doxorubicin in a mouse not treated withtoremifene. The bottom two curves show tumors that were treated withtoremifene. Clearly, toremifene appears to sensitize these MDR MDA A-1tumors to doxorubicin.

Discussion:

These results suggest that the high achievable tumor toremifeneconcentrations seen with topical toremifene in vivo, the greater thesynergistic effects will be at reversing the acquired drug resistance todoxorubicin.

EXPERIMENT 4

Methods

Topical toremifene therapy of a 15-year old female baboon with aspontaneous non-Hodgkin's lymphoma was evaluated. Enlarged lymph nodeswith diameters of 1-2 cm were evident in the both groins. Toremifene wasdissolved in a. mixture of dimethylsulfoxide, ethanol andmethylcellulose. Over a period of 8 days, a total of 1.4 g toremifenewas applied primarily on the right tumor region. Following 15 days fromthe start of topical therapy, an oral treatment with 1 g of toremifeneon days 15, 17, 19 and 23 was instituted, with a total dose of 4 g. Onday 25 following start of treatment, topical therapy of right groin wasre-instituted while continuing the oral treatment. During that period, atotal of 1.4 g of toremifene was administered transdermally and 11 gorally. The response to therapy was determined by measuring the sizes ofthe tumors on two perpendicular dimensions.

Results

The response to the therapy is given in FIG. 3a and 3b. A clear responsewas observed in the treated right inguinal lymphoma whereas theuntreated left lymphoma increased in size during the topical treatmentperiod. During the oral toremifene therapy the left inguinal lymphomastabilized, whereas the right inguinal lymphoma begun to regrow.Consequently, topical toremifene therapy was reinstituted on this tumor.Similarly to the initial response to topical therapy, the lymphomaregressed from tumor area of 476 mm² to 24 mm².

The treatment of a regional T-cell lymphoma in a female baboon showedthat topical toremifene can produce an in vivo antitumor response.

EXPERIMENT 5

Methods

Toremifene in the form of toremifene citrate was first dissolved inDMSO, after which it was added to 3% methyl cellulose in ethanol to afinal concentration of 1 mg/ml.

Six male Monodelphis domestica were given toremifene topically for 5days. The dose was 0.5 mg/d in 3 and 1 mg/d in 3 animals. Toremifene wasapplied topically to shaved skin on a surface area of 1 cm² of the lowerback. At day 5 the animals were sacrificed, and the concentrations oftoremifene and its metabolites were measured in plasma, skin, testicles,liver, eyes, brains and heart.

Three human melanoma cell lines (TD 36, TD 30A and SK-MEL-31) and threeexperimental cell lines (TD 1.4, TD 7.2 and TD 8) derived fromUVB-induced melanocytic nevi in Monodelphis domestica were grown invitro to assess the cytostatic IC-50 values for toremifene. Themetastatic melanoma cell line, SK-MEL-31, was obtained from the AmericanType Culture Collection (Rockville, Md.). Two additional human celllines were derived from portions of surgigally-excised large diameter,superficial spreading melanomas. TD 1.4 was developed from a benignmelanoma, whereas TD 7.2 and TD 8 were derived from melanocytichyperplasias. IC-50 values were determined from semi-log plots ofpercent cell survival vs. concentration of toremifene citrate.

Results

The mean plasma and tissue concentrations of toremifene in 6 animalsfollowing topical administration of 0.5 and 1 mg/day for 5 days aregiven in Table 6. Toremifene concentration in skin was more than500-fold as compared to other tissues.

The 50% growth inhibitory concentrations (IC-50) of the human andexperimental cell lines for toremifene is shown in Table 7. Growthinhibitory effect of toremifene was very similar in all six cell linesof melanocyte origin. The IC-50s had a range of 5.8-9.6μM.

Discussion

In the experiment we demonstrated that toremifene has a clear cytostaticeffect on human and experimental melanomatous cell lines in vitro.Although a cytostatic effect was observed in all cell lines, theconcentrations needed to produce this effect were relatively high.However, topical administration of toremifene produces skinconcentrations that are far beyond these in vitro concentrations,suggesting that a clear cytostatic effect could be obtained on cutaneousmelanoma in vivo.

                  TABLE 1                                                         ______________________________________                                        Toremifene concentrations following 5 days of IP vs.                          topical treatment with toremifene                                             Tissue IP           Topical      Topical/IP                                   ______________________________________                                        Tumor  7.08         1087.34      153.58                                       (μg/g)                                                                            (3.55-11.86)  (23.47-1924.77)                                          Serum  4.37         1.00         0.23                                         (μg/ml)                                                                            (0.1-12.79) (0.46-1.84)                                               Brain  1.85         1.45         0.78                                         (μg/g)                                                                            (1.17-2.20)  (1.29-1.61)                                               Uterus 29.97        2.94         0.10                                         (μg/g)                                                                            (26.47-35.90)                                                                              (2.33-3.53)                                               Liver  14.25        4.21         0.30                                         (μg/g)                                                                            (11.2-18.60) (3.46-5.62)                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        N-demethyltoremifene concentrations following IP vs.                          topical treatment                                                             Tissue   IP          Topical    Topical/IP                                    ______________________________________                                        Tumor    1.27        8.43       6.64                                          (μg/g)                                                                              (0.88-1.74)  (1.15-13.86)                                            Serum    0.18        0.08       0.44                                          (μg/ml)                                                                             (0.10-0.30) (0.06-0.09)                                              Brain    0.49        0.29       0.59                                          (μg/g)                                                                              (0.26-0.73) (0.0-0.49)                                               Uterus   1.18        0.56       0.47                                          (μg/g)                                                                              (1.02-1.51) (0.38-0.76)                                              Liver    0.94        0.92       0.98                                          (μg/g)                                                                              (0.92-0.98) (0.80-1.10)                                              ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Trans 4-hydroxytoremifene concentrations following IP                         vs. topical treatment                                                         Tissue   IP           Topical   Topical/IP                                    ______________________________________                                        Tumor    0.88         2.33      2.65                                          (μg/g)                                                                              (0.64-1.06)  (0.82-3.26)                                             Serum    0.16         0.10      0.63                                          (μg/ml)                                                                             (0.12-0.20)  (0.04-0.17)                                             Brain    0.35         0.35      1.00                                          (μg/g)                                                                              0.22-0.43)   (0.31-0.40)                                             Uterus   0.83         0.70      0.84                                          (μg/g)                                                                              (0.46-1.05)  (0.50-0.80)                                             Liver    0.60         0.98      1.63                                          (μg/g)                                                                              (0.56-0.66)  (0.51-1.43)                                             ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Comparison of cellular concentrations of toremifene                           following in vitro exposure, intraperitoneal injection and                    topical administration                                                        Sample     Route of Administration                                                                       Toremifene Conc.                                   ______________________________________                                        In Vitro (Cell                                                                           Direct Application                                                                            0.149 μg/mg                                     pellets)                                                                      In Vivo    Intraperitoneal Injection                                                                     0.007 μg/mg                                     (Tumor)                                                                       In Vivo    Topical Administration                                                                        1.087 μg/mg                                     (Tumor)                                                                       ______________________________________                                         Concentrations are average of three determinations.                      

                  TABLE 5                                                         ______________________________________                                        Toremifene concentrations following a single dose IP                          vs. topical treatment toremifene (500 μg)                                  in DMSO or methylcellulose                                                                                       DMSO/  MC/                                 Tissue                                                                              IP         DMSO      MC      IP     IP                                  ______________________________________                                        Tumor 2.57       4.65      1.80    1.81   0.70                                (μg/g)                                                                           (1.64-4.46)                                                                               (3.41-6.96)                                                                            (0.95-3.10)                                        Serum 0.09       0.03      0.02    0.33   0.22                                (μg/                                                                             (0.04-0.14)                                                                              (0.004-0.04)                                                                            (0.00-0.04)                                        ml)                                                                           Uterus                                                                              23.60      0.92      1.10    0.04   0.05                                (μg/g)                                                                           (13.58-40.17)                                                                             (0.66-1.35)                                                                            (0.64-1.92)                                        ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        The mean concentration of toremifene in plasma                                (nmol/ml) and various tissues (nmol/g) following topical                      toremifene administration of 0.5 and 1 mg/day for 5 days in                   6 male Monodelphi domestica.                                                           0.5 mg/day 1 mg/day                                                  ______________________________________                                        Plasma     0            0                                                     Skin       1296.40 ± 1003.93                                                                       1237.35 ± 768.90                                   Liver      1.07 ± 0.69                                                                             3.60 ± 2.23                                        Testicles  1.52 ± 2.13                                                                             0.52 ± 0.05                                        Brain      0.82 ± 0.99                                                                             0.82 ± 0.62                                        Eyes       0            0.17 ± 0.05                                        Heart      0            0.21 ± 0.16                                        ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        IC-50 values of the human and experimental melanomatous                       cell lines for toremifene                                                                  Cell line                                                                              IC-50 (μM)                                           ______________________________________                                        Human          TD 36      5.8                                                                TD 30 A    7.7                                                                SD-MEL-31  8.1                                                 Experimental   TD 8       7.7                                                                TD 1.4     9.3                                                                TD 7.2     9.6                                                 ______________________________________                                    

We claim:
 1. A transdermal preparation comprising as active ingredienttoremifene or one of its metabolitesN-demethyltoremifene(4-chloro-1,2-diphenyl-[4-[2-(N-methylamino)ethoxy]phenyl]-1-butene)or 4-hydroxytoremifene(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-[4-[2-(N,N-dimethylamino)ethoxy]phenyl]-1-butene) or a pharmaceuticallyacceptable non-toxic salt thereof, together with one or morepharmaceutically acceptable excipients.
 2. A preparation according toclaim 1 which is in the form of an ointment, emulsion, lotion, solution,gel or cream.
 3. A preparation according to claim 1 where thepreparation is a transdermal delivery patch system.
 4. A preparationaccording to claim 2 where the active ingredient is in the form of acomplex of a cyclodextrin.
 5. A preparation according to claim 1 wherethe active ingredient is toremifene or a pharmaceutically acceptablesalt thereof.
 6. A transdermal preparation according to claiam 1 for usein the treatment of breast cancer.
 7. A transdermal preparationaccording to claim 1 for use in the adjuvant therapy of breast cancer.8. A transdermal preparation according to claim 1 for use in thereversal of multidrug resistance of cancer cells to cytotoxic drugs. 9.A transdermal preparation according to claim 1 for use in the treatmentof melanoma, lymphoma, Kaposi's sarcoma or fungoides mycosis.
 10. Amethod of treatment of breast cancer comprising administeringtransdermally to a subject in need of such treatment an effective amountof a transdermal preparation comprising as active incredient toremifeneor one of its metabolitesN-demethyl-toremifene(4-chloro-1,2-diphenyl-[4-[2-(N-methylamino)ethoxy]-phenyl]-1-butene)or4-hydroxytoremifene(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-[4-[2-(N,N-dimethylamino)ethoxy]-phenyl]-1-butene)or a pharmaceutically acceptable non-toxic salt thereof, together withone or more pharmaceutically acceptable excipients.
 11. A method forreversing the multidrug resistance of cancer cells to a cytotoxic drugcomprising administering transdermally a multidrug resistance reversaleffective amount of toremifene or one of its metabolitesN-demethyltoremifene(4-chloro-1,2-diphenyl-[4-[2-(N-methylamino)ethoxy]-phenyl]-1-butene)or4-hydroxytoremifene(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-[4-[2-(N,N-dimethylamino)-ethoxy]phenyl]-1-butene)or a pharmaceutically acceptable non-toxic salt thereof, together withone or more pharmaceutically acceptable excipients, to a subject in needof such treatment.
 12. A method of treatment of melanoma, lymphoma,Kaposi's sarcoma or fungoides mycosis comprising administeringtransdermally to a subject in need of such treatment an effective amountof a transdermal preparation comprising as active incredient toremifeneor one of its metabolitesN-demethyltoremifene(4-chloro-1,2-diphenyl-[4-[2-(N-methylamino)ethoxy]-phenyl]-1-butene)or4-hydroxytoremifene(4-chloro-1-(4-hydroxyphenyl)-2-phenyl-1-[4-[2-(N,N-dimethyl-amino)ethoxy]phenyl]-1-butene)or a pharmaceutically acceptable non-toxic salt thereof, together withone or more pharmaceutically acceptable excipients.
 13. A preparationaccording to claim 3 where the active ingredient is in the form of acomplex of a cyclodextrin.
 14. A preparation according to claim 2 wherethe active ingredient is toremifene or a pharmaceutically acceptablesalt thereof.
 15. A preparation according to claim 3 where the activeingredient is toremifene or a pharmaceutically acceptable salt thereof.16. A preparation according to claim 4 where the active ingredient istoremifene or a pharmaceutically acceptable salt thereof.
 17. Atransdermal preparation according to claim 2 for use in the treatment ofbreast cancer.
 18. A transdermal preparation according to claim 3 foruse in the treatment of breast cancer.
 19. A transdermal preparationaccording to claim 4 for use in the treatment of breast cancer.
 20. Atransdermal preparation according to claim 5 for use in the treatment ofbreast cancer.
 21. A transdermal preparation according to claim 2 foruse in the reversal of multidrug resistance of cancer cells to cytotoxicdrugs.